1. Field of the Invention
The present invention relates to a dry immunoassay element in which a homogeneous enzyme immunoassay is utilized, and an immunoassay process in which the dry immunoassay element is used.
Analyses of the constituents originated from the living body or chemicals contained in the body fluids, such as blood and urine, are useful for diagnosing the condition of diseases or judging the developement of curing, and thus they occupy important parts in the field of clinical test. The so-called enzyme immunoassay has been known in the art as one of the methods for analyzing such a constituent (ligand) generally present in a small amount in the body fluids. The enzyme immunoassay may be classified into heterogeneous systems for which B/F (Bound/Free) separation must be effected, and homogeneous system for which B/F separation is not necessary. The reactions in the homogeneous system is based on the phenomenon that the enzymatic activity of the labelling enzyme is affected by some interference caused by binding of an antibody to the antigen (ligand), and the inhibition due to antigen-antibody binding is generally utilized. It is considered that the enzymatic activity is suppressed by a steric hindrance for binding the enzyme to the substrate or a change in three-dimensional structure of the enzyme, when the antibody which is generally a large molecule is bound to the antigen in the enzyme-labelled antigen. Meanwhile, in the routine clinical tests in which a number of test samples are to be handled, it is demanded that the individual samples should be analyzed rapidly by simple operations, more desirously by automated operation sequence.
2. Prior Art Statement
To comply with the demand, dry analysis elements have been proposed (see, for example, Unexamined Japanese Patent Publication Nos. 53888/1974 (corresponding to U.S. Pat. No. 3,992,158), 77356/1984 (corresponding to EP 0 097952A) and 102388/1984 and U.S. Pat. No. 4,459,358.)
A dry analysis element has been known, in which a homogeneous enzyme immunological reaction is utilized (see Unexamined Japanese Patent Publication No. 32136/1989 (corresponding to EP 0347839A)). This known dry analysis element comprises the following three ingredients in the same or other layers in a composite multi-layered structure:
(A) An antigen having a high molecular weight (a coupling product of a ligand or a derivative thereof with a high molecular weight compound; hereinafter referred to as "polymerized antigen"); PA1 (B) A water-insoluble high polymer substrate; and PA1 (C) A conjugate of an antibody against the ligand and an enzyme for the substrate. PA1 (a) applying said sample into a substrate layer containing a non-diffusible substrate which forms a diffusible material in the presence of said enzyme; PA1 (b) allowing to migrate said diffusible material formed in said substrate layer into a reagent layer containing a fragmenting enzyme for further decomposing to a lower molecular weihgt product; and PA1 (c) measuring the amount of said lower molecular weight product formed in said reagent layer.
The antigen supplied by spotting onto the analysis element binds to the antibody-enzyme conjugate through a competitive reaction with the reaction of the polymerized antigen. The complex of antigen-antibody-enzyme reacts with the water-insoluble high polymer substrate to form a soluble lower molecular weight product. On the other hand, the complex of polymerized antigen-antibody-enzyme formed by the binding with the polymerized antigen cannot exhibit the enzymatic activity to the high polymer substrate. Accordingly, as the quantity of the antigen in the sample is increased, the product produced by the enzymatic reaction increases. This product is allowed to diffuse into a detection layer where the quantity of the product is determined by measuring the optical density of an absorption resulted by the colored chemical group, to make it possible to analyze the antigen in the sample quantitatively.
However, in this known analysis element, since a high polymer substrate bound to a dye, such as dye-starch, is used and the dye bound to amylose which is the decomposition product formed by the action of amylase is determined, the high polymer substrate and the reaction product must be separated prior to the measurement or determination. It is, therefore, necessary to provide a light-shielding layer containing, for example, titanium dioxide fine particles between the reagent layer containing the unreacted substrate and the detection layer for receiving the reaction product. The analysis element having such a laminated structure is not preferred because it is necessary to take some time for the soluble reaction product formed in the reagent layer to be diffused into the detection layer, leading to the result that rapid quantitative determination, which is a major merit of the dry analysis element, cannot be carried out.
It is possible to improve the diffusibility of the reaction product by introducing a hydrophilic group, such as carboxyl or sulfo group, into the substrate to accelerate the diffusion of the reaction product. However, the site on which such a substituting group can be introduced is limited, and the introduction thereof might cause a demerit that the molecular extinction coefficient of the dye site governing the sensitivity of the analysis is lowered.